Noncatalytic Assembly of Ribonuclease III with Double-Stranded RNA Jaroslaw Blaszczyk, Jianhua Gan, Joseph E Tropea, Donald L Court, David S Waugh, Xinhua Ji  Structure  Volume 12, Issue 3, Pages 457-466 (March 2004) DOI: 10.1016/j.str.2004.02.004

Figure 1 RNase III Proteins and Sequences (A) Representatives of RNase III proteins: Hs-Dicer (1924 amino acid residues, GenBank AB028449), Dm-Drosha (1327 amino acid residues, SWISS-PROT Q9XYN5), Sc-Rnt1p (471 amino acid residues, SWISS-PROT Q02555), and Ec-RNase III (226 amino acid residues, SWISS-PROT P05797). The scale on top indicates the lengths of polypeptide chains. Cyan box represents helicase domain, green box PAZ domain, red box endoND, and blue box dsRBD. Additional domain structures in Hs-Dicer and Dm-Drosha are not shown. (B) Sequence alignment of Aa-RNase III (SWISS-PROT O67082), Tm-RNase III (SWISS-PROT Q9X0I6), and Ec-RNase III (SWISS-PROT P05797). Secondary structural elements are indicated with shading, helices in green and strands in blue. Boxed amino acid residues at the C termini are not observed. Underlined is the RNase III signature motif. Shaded in red are seven Ec-RNase III residues for which data is available for ten mutant proteins, including (1) G44D (rnc105 [Bardwell et al., 1989; Nashimoto and Uchida, 1985]); (2) I47N (rnc−; H.K. Peters, N. Costantino, and D.L.C., unpublished data); (3) G97E (rnc97 [Davidov et al., 1993]); (4) E117K (rnc70 [Dasgupta et al., 1998; Inada et al., 1989; Li and Nicholson, 1996]), E117A (Li and Nicholson, 1996), and E117Q and E117D (Sun and Nicholson, 2001); (5) Q153P (rnc10 [Inada and Nakamura, 1995]); (6) D155E (rnc7 [Inada and Nakamura, 1995]), and (7) A211V (rev3 [Nashimoto and Uchida, 1985]). Structure 2004 12, 457-466DOI: (10.1016/j.str.2004.02.004)

Figure 2 Impact of E110K Mutation on the Mg2+ Binding Site of Aa-RNase III (A) Stereoview of the final 2Fo-Fc electron density contoured at 1σ (green net) for the Aa-E110K mutation site. Residues E40, D44, D107, and K110, and two water molecules are illustrated as ball-and-stick models in atomic colors (gray carbon, blue nitrogen, and red oxygen). (B) Stereoview showing the superposition of Aa-endoND·Mg2+ (in cyan) and Aa-E110K·dsRNA (in atomic colors). The coordination bonds of Mg2+ are shown as solid lines and hydrogen bonds as dashed lines. Structure 2004 12, 457-466DOI: (10.1016/j.str.2004.02.004)

Figure 3 Overall Structure of Aa-E110K·dsRNA (A) Illustration of a biological dimer of the Aa-E110K·dsRNA complex. The crystallographically independent molecule and its symmetry mate are indicated by N-C, and N′-C′, respectively. Secondary structural elements are labeled for those in dsRBD and α3 in the endoND. The endoND, dsRBD, dsRNA, endoNDsym, dsRBDsym, and dsRNAsym are colored yellow, green, green, cyan, blue, and blue, respectively. The RNase III signature motif at the N terminus of α3 is highlighted in red. Helices, β strands and loops are drawn as spirals, arrows, and pipes, respectively. (B) A different view of dimeric Aa-E110K·dsRNA related to the view in (A) by a 90° rotation around the vertical axis. Structure 2004 12, 457-466DOI: (10.1016/j.str.2004.02.004)

Figure 4 The dsRBD-dsRNA Interaction (A) Stereoview showing the superposition of the Aa-dsRBD·dsRNA portion (in orange) of Aa-E110K·dsRNA (this work) with Xl-dsRBD·dsRNA (in cyan [Ryter and Schultz, 1998]) on the basis of dsRBD Cα alignment. (B) Stereoview illustrating the recognition of nucleotide base functional groups by dsRBD residues. Aa-E110K residues Q157, Q161, and H179 (in orange) correspond to Xl-dsRBD residues Q118, V122, and P140 (in cyan), respectively. Structure 2004 12, 457-466DOI: (10.1016/j.str.2004.02.004)

Figure 5 Comparison of Full-Length RNase III Model and Structures (A) The crystal structure of Aa-E110K·dsRNA (this work). (B) The model of Aa-RNase III·dsRNA (Blaszczyk et al., 2001). The dsRNA is illustrated as vdw space-filling models in atomic color scheme (carbon in light gray, nitrogen in blue, and oxygen in red). One Aa-E110K chain of the dimer is colored in orange and the other in cyan, and the dsRBD is highlighted with dark gray surfaces. (C) The alignment, using endoND Cα positions, of the crystal structure of Aa-E110K·dsRNA (in cyan; this work), the model of Aa-RNase III·dsRNA (in orange [Blaszczyk et al., 2001]) and the crystal structure of ligand-free Tm-RNase III (in red, PDB entry 1O0W). The dsRNA is not shown for clarity. The molecular orientation is related to those in (A) and (B) by a 90° rotation around the horizontal axis for an optimal view of the flexible linker between the endoND and dsRBD. Structure 2004 12, 457-466DOI: (10.1016/j.str.2004.02.004)